Compositions and methods for inhibiting nematocyst discharge

ABSTRACT

Compositions for inhibiting nematocyst or polar capsule discharge are provided in the form of an effective amount of antihistamine, an effective amount of antihistamine and an effective amount of at least one cation, or an effective amount of at least one cation. The compositions can be in the form of ointments or can be added to the environment surrounding the nematocysts or polar capsules. Methods of inhibiting nematocyst or polar capsule discharge using the compositions of the invention are also provided.

RELATED APPLICATION

This application is a continuation-in-part of U.S patent applicationSer. No. 08/865,332, filed May 29, 1997, now U.S. Pat. No. 6,132,747.

TECHNICAL FIELD

This invention is in the field of compositions which repel and preventthe stinging of marine organisms, such as jellyfish. In particular,compositions and methods for inhibiting the discharge of the stingingstructure (nematocyst or polar capsule) are provided.

BACKGROUND

Swimmers and divers throughout the world are familiar with the stingingorganisms of the oceans such as jellyfish, sea anemone and coral.Jellyfish stings, although seldom fatal, are a major public healthproblem. Lotan et al. (1992) Marine Biology 112:237-242; Lotan et al.(1994) Marine Ecology Progress Series 109:59-65. In the summer months,it is estimated that over 500,000 swimmers and divers in the ChesapeakeBay area and 200,00 persons in Florida are stung by jellyfish, mainly by“sea nettles,” the common name of Chrysaora quinquecirrha. Burnett etal. (1992) MMJ 41(6):509-513. Similarly, between the months of March andAugust, one in four Florida bathers are stung and more than 10,000persons require emergency medical treatment for pruritic eruptionscaused by contact with jellyfish larvae known as “sea lice.” Tomchik etal. (1993) JAMA 269(13):1669-1672.

The members of the phylum Cnidaria (e.g., jellyfish, sea anemone andcoral) and the phylum Myxozoa are all equipped with stinging subcellularorganelles, known as nematocysts, cnidocysts, or polar capsules. Thenematocysts are located in specialized cells (nematocytes) and consistof a capsule containing a condensed tubule with potent toxins andthreads. When nematocysts discharge, the tubule penetrates into itstarget organism and releases its toxins. The threads arrayed on thetubule enhance the anchoring and attachment of the nematocyst tubule toits target. Thus, nematocysts are involved in target recognition, toxindelivery, infection and attachment.

All members of the phyla Cnidaria and Myxozoa contain nematocysts ofvarying sizes, shapes and types. Mariscal, pp. 129-178, “Nematocysts” inCOELENTERATE BIOLOGY: REVIEWS AND NEW PERSPECTIVES, eds. Muscatine andLenhoff (Academic Press, New York, 1974). These different types ofcnidocysts function in diverse biological roles including capture ofprey, toxin delivery, recognition, attachment, adherence and infection.(see, e.g., Tardent (1995); Bioessays 17(4):351-362; Lotan et al. (1995)Nature 375:456; Lotan et al. (1996) Expt'l Zool. 275:444-451; Lotanetal., pp. 132-144, “Toxicology and ecology and the Mediterraneanjellyfish Rhopilema nomadica” in BIOCHEMICAL ASPECTS OF MARINEPHARMACOLOGY, eds. Lazarovici et al. (Alaken, Inc., Fort Collins, Colo.,1996); Spaulding (1972) Biol. Bull. 143:440-453; Holstein and Tardent(1984) Science 223:830-833 and Mariscal, supra. Thus, although bestknown for their stinging capabilities, nematocytes also play a key rolein recognition, attachment and infection. For example, parasites fromthe phylum Myxozoa use nematocysts (polar capsules) to recognize andinfect their hosts. El-Matbouli et al. (1995) J. Fish Biol. 46:919-935and Yokoyama et al. (1995) Diseases Aquatic Organ. 21:7-11.

The main body of the nematocyte cell consists of a dense capsule, thenematocyst, within which is a high folded eversible tubule. Discharge(eversion) of this tubule is driven by the build up of a high internalhydrostatic pressure of approximately 150 atmospheres within thecapsule. The eversion of the internally folded tubule occurs within 3microseconds at accelerations of up to 40,000×g, one of the most rapidmechanical events in cell biology.

The nematocyte can be sub-divided into 3 morphological compartments withdifferent functional entities: the capsule lumen and wall, the tubuleand the sensory organelles. The capsule wall and lumen are the maincomponents involved in developing the driving force for nematocystdischarge. The tension on the inner capsule wall during nematocystdischarge reaches up to 375 MPa. Holstein (1994) Science 265:402-404.The strong capsule wall is highly permeable to water with a pore size of600 Dalton. Within the resting nematocyst capsule, concentrations of upto 0.5 M of cations such as Ca⁺⁺, Mg⁺⁺ or K⁺ can be found. (see, e.g.,Tardent, supra; Lubbock et al. (1981) PNAS 78(6):3624-3628; Godknecht etal. (1988) Marine Biology 100:83-92; Lubbock and Amos (1981) Nature290(5806):500-501; Weber (1989) Int. J. Biochem. 184:465-476; Hidaka(1993) Biol. Bull. 184:97-104 and Gerke (1991) Hydrobiologia216/217:661-669 for discussions of cations and nematocysts). The anioniccounterparts are represented by poly-γ-L-glutamatic acid (PGA) invarying degrees of polymerization. During nematocyst discharge, anextreme increase in internal capsule osmotic pressure occurs due to theinflux of water. It has been suggested that the influx of water into thecapsule is mediated by an internal release of the cations, such as Ca⁺⁺in sea anemone or K⁺ in hydra normally combined with PGA. This osmoticpressure is translated into hydrostatic pressure causing the eruptionand then evagination of the tubule form the nematocyst capsule(discharge). After the nematocyst discharge the internal cationconcentration of the capsule is dissolved into the surrounded fluids.

The second compartment of the nematocyst is a highly condensed eversibletubule. This tubule serves the main role in nematocyte biologicalfunction; namely, the interaction or delivery of substances from thecnidarian or myxozoan into its target. The tubule, which is 200-850 μmwhen elongated, is twisted more than a hundred times around its axis andis packed into the 3-10 μm diameter of the nematocyst. Godknecht &Tardent (1988) Marine Biol. 100:83-92. Hollow barbs, arrayed on theinner surface of the tubule, become everted during discharge and play animportant role in the penetration and anchoring of the tubule into itsprey. Toxins, contained on the outer surface before discharge, aredelivered through the barbs after the nematocyst is anchored. Lotan etal. (1995), supra.

The sensory systems of the nematocyte are responsible for the control ofnematocyst discharge. Discharge of nematocysts requires both chemicaland mechanical stimulation of the sensory organelles. In the sea anemoneAiptasia pallida, two classes of chemical receptors have beenidentified. One type of receptor is triggered by N-acetylated sugars,while the second chemoreceptor is triggered by certain amino acids.Thorington et al. (1988) Biol. Bull. 174:163-171. The mechanical sensorysystem is dependent on activation of the chemoreceptors. Thesemechanoreceptors, however, can be adjusted by the organism, forinstance, by tuning them to unique frequency signals emitted by afavored prey.

Certain fish diseases, including whirling disease, are caused byinfection with an obligate parasite of the phylum myxozoan. (see e.g.,for descriptions of myxozoan, Yokoyama et al. (1993) Dis. Aquat. Org.17:223-228; Yokoyama et al. (1995) Dis. Aquat. Org. 21:7-11; Smothers etal. (1994) Science 265:1719-172; and El-Matbouli et al. (1995) J. FishBiology 46:919-935). These parasites require two hosts, a fish and anaquatic annelid. The stages emerging from each host are infectious onlyfor the other host, however, at each infectious stage, activated polarcapsules (nematocyst) release a tubule that penetrates or adheres to theintegument of the target. Host recognition is species-specific,indicating that nematocysts serve an important role in recognition oftheir host (El-Matbouli, supra).

In sum, nematocysts provide an effective method of delivering asubstance deep into the target. Because nematocysts are able topenetrate their target so efficiently, it is difficult to remove them,or to treat after the toxin has penetrated. Conventionally, nematocystsstings have been treated with antidotes such as steroids, aluminumsulfate/surfactant and antihistamines. Tomchik, supra. For example,Wasuwat (1970) Nature 225:758 describes how Thai fisherman use anextract made from the leaves of Ipomoea pea-caprae as an antidote tojellyfish poison. When the extract was analyzed, it was found to bemildly antihistaminic. The extract exhibits the same effect againstjellyfish poison as two commercially prepared antidotes containingantihistamines. In Australia, vinegar is recommended for treatment oflethal box jellyfish stings. Hartwick et al. (1980) Med. J. Aust.1:15-20. Vinegar is not recommended for stings by other species. Fenneret al. (1993) Med. J. Aust. 158:498-501.

Since post-sting treatments for nematocyst stings are oftenunsatisfactory, the search for ways to prevent nematocyst discharge hasbeen ongoing. The most often prescribed method of preventing jellyfishstings is avoiding any contact with the nematocysts. Tomchik, supra.However, in the case of microscopic larvae, this often means foregoingall ocean activities during the months of high incidence, (e.g., Marchthrough August in Florida). It would, therefore, be useful to have ameans for inhibiting nematocyst discharge even when contact does occur.

Australian patent application 67563/94 (WO 94/17779) discloses topicalhydrodispersion preparations that are reported to be effective inpreventing nematocyst discharge as measured by scanning electronmicroscopy (SEM). The formulations contain inorganic micropigmentsincorporated into the lipid phase of the hydrodispersion; an optional UVfiltering substance and are essentially free of emulsifiers.

Lubbock (1979) J. Exp. Biol. 83:283-292 describes how proteinaceouscompounds tend to induce a stronger response leading to nematocystdischarge in sea anemones than either polysaccharides or lipids. Theauthors could determine no simple recognition basis and speculated thatthe process of nematocyst discharge was complex. Lubbock and Amos,supra, disclose that isolated nematocyst capsules do not discharge in 50mM CaCl₂. The authors report that inhibition of nematocyst dischargeoccurs only if a solute that could not rapidly penetrate the capsulewall is used, for example, high molecular weight polyethylene glycol.Thus, calcium in the surrounding environment may stabilize nematocystsbecause it reduces the differential between the calcium concentrationoutside the capsule and inside the capsule. Normally, the calciumconcentration inside the nematocyst capsule is approximately 600 mM.Normal calcium concentration in sea water is around 7 mM, about 100 foldless than inside the capsule. Thus, increasing the calcium concentrationoutside the capsule to 50 mM reduces the differential to around 10 foldand may be involved in inhibiting nematocyst discharge.

Heeger et al. (1992) Marine Biology 113:669-678 tested the ability ofthree commercially available sunscreen lotions to inhibit jellyfishnematocyst discharge on samples of live human skin. Two of the threelotions were effective at reducing the number of nematocysts discharged.The authors concluded that glycerol and oil components of the lotionscould be masking or suppressing the effects of natural stimuli of theskin, however, even the lotion which did not inhibit nematocystdischarge contained these substances. Hartwick et al. (1980) Med. J.Australia 1:15-20 report that commercial sting remedies provide do notinhibit nematocyst discharge.

Thorington et al. Biol. Bull. 174:163-171 (1988) describe two classes ofnematocyst chemoreceptor. One class of chemoreceptors is specific forN-acetylated sugars, and is unaffected by antihistamines. The secondclass of chemoreceptors is specific for amino acids and is inhibited byantihistamines. The authors hypothesized that the N-acetylated sugarreceptors are the initial trigger for nematocyst discharge and that theantihistamine-affected receptors are probably activated only uponleakage of amino acids from the puncture wound created in the prey bythe sugar-triggered nematocyst. It is therefore, unexpected that thepresent invention has shown that antihistamines alone are effective atinhibiting nematocyst discharge.

The present invention provides novel compositions that effectivelyinhibit nematocyst discharge. The compositions described herein areextremely effective at inhibiting nematocyst discharge when appliedtopically prior to exposure to nematocysts. In addition, it was thesurprising discovery of the inventor that placing antihistamines and/orcations into the aqueous environment surrounding nematocyst-bearingorganisms effectively inhibits nematocyst discharge. Thus, the presentinvention provides both protection for swimmers, divers and fishermen,as well as protection from nematocyte-facilitated infection in contextssuch as fish farms where the surrounding environment can be treated.

DISCLOSURE OF THE INVENTION

In accordance with one embodiment of the present invention, acomposition for inhibiting the discharge of nematocysts comprising aneffective amount of an antihistamine is provided. In accordance withanother embodiment of the present invention, compositions for inhibitingthe discharge of nematocysts comprising an effective amount of at leastone cation are provided. In accordance with yet another embodiment ofthe present invention, compositions for inhibiting the discharge ofnematocysts comprising effective amounts of an antihistamine and atleast one cation are also provided.

The nematocysts may be discharged from organisms selected from the groupconsisting of Hydrozoa, Anthozoa, Myxozoa and Schyphoza, preferably fromthe group consisting of Aurelia sp., Pelagia sp., Chrysaora sp.,Anthoplaura sp, Rhopilema sp., Physalia sp., Cyanea sp., Linuche sp.,Catostylus, Carybdea sp., Chironex sp., Stomolophus sp., Rhiozostoma andCorinactis sp., more preferably from the group consisting of Aureliaaurita, Corynactic californica, Anthopleura elegantissima, Pelagianoctiluca., Chrysaora quinquecirrha and Anthoplaura sp

In one embodiment, the antihistamine is present in a concentration offrom about 0.0005% to about 2.0%, preferably from about 0.001% to about0.2%. The antihistamine can be any antihistamine, preferablydiphenhydramine, cimetidine or tripelennamine, more preferablydiphenhydramine.

In yet a further embodiment, the composition comprising an antihistaminefurther comprises at least one cation. The cation can be Ca⁺⁺, K⁺, Na⁺,Mn⁺⁺, Co⁺⁺, Mg⁺⁺, or Fe⁺⁺ and is present in a concentration of fromabout 50 mM to about 1M, preferably from about 50 mM to about 500 mM,more preferably from about 50 mM to about 200 mM.

In another embodiment, the composition comprises at least one cation.The cation can be Ca⁺⁺, K⁺, Na⁺, Mn⁺⁺, Co⁺⁺, Mg⁺⁺, or Fe⁺⁺ and ispresent in a concentration of from about 50 mM to about 1M, preferablyfrom about 50 mM to about 500 mM, more preferably from about 50 mM toabout 200 mM.

In another embodiment, compositions within the present invention areincorporated into an ointment base. In a preferred embodiment, theointment base does not comprise a glycoprotein, and is a lipid or asilicone polymer. In yet a further embodiment, the ointment base furthercomprises a sunscreen.

In yet a further embodiment of the present invention, methods areprovided for inhibiting nematocyst discharge comprising applyinginventive compositions within the present invention to the skin of asubject prior to contact with nematocysts. In another embodiment, methodare provided for inhibiting nematocyst discharge in an environmentsurrounding the nematocyst.

As will become apparent, preferred features and characteristics of oneaspect of the invention are applicable to any other aspect of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing three steps activation ofnematocysts. Chemical secretions of stimulants from the skin bind tosensors. Signals from the sensors activate the capsule. High internalpressure builds up in the capsule and leads to ejection of the tubule.

FIG. 2 is a schematic diagram depicting how the formulation will preventor inhibit discharge of nematocysts. In this depiction, a topicalointment containing the inventive composition is applied to the skin.Secretions of stimulants from the skin are thereby reduced or prevented.The sensors and capsule discharge mechanism are chemically inactivatedby the inventive composition.

MODES FOR CARRYING OUT THE INVENTION

Throughout this application, various publications, patents, andpublished patent applications are referred to by an identifyingcitation. The disclosures of the publications, patents, and publishedpatent specifications referenced in this application are herebyincorporated by reference into the present disclosure to more fullydescribe the state of the art to which this invention pertains.

As used herein, certain terms will have defined meanings.

The term “nematocyst” or “cnidocyst” or “polar capsule” is used in theconventional sense to refer to the subcellular stinging structures foundin the nematocytes or cnidocytes. Nematocysts are described in detail inTardent, supra and Mariscal, supra. These stinging organelles arecapable of penetrating, anchoring into and injecting a substance such asa toxin into a target organism. The terms “nematocyte” or “cnidocyte” or“cnidoblast” are intended to refer to the specialized cell which containthe nematocysts. Non-limiting examples of classes of the phylum Cnidariawhich possess cnidocytes include, for example, Hydrozoa, Anthozoa,Myxozoa and Scyphozoa. Examples of jellyfish include, but are notlimited to, Aurelia sp., Pelagia sp., Chrysaora sp., Anthoplaura sp.,Rhopilema sp., Physalia sp., Cyanea sp., Linuche sp., Catostylus sp.,Carybdea sp., Chironex sp., Stomolophus sp., Rhiozostoma sp., Corinactissp. and the like.

The term “antihistamine” refers to the general class of compounds whichinhibit histamine binding. There are generally two types of histaminereceptors, H₁ and H₂. Antagonists of both types of histamine receptorsare effective at decreasing or preventing nematocyst discharge.

The term “cation” refers to any positively charged ion. The term “cationsource” refers to any substance which is capable of supplying positivelycharged ions. Preferably, the cations are metal cations. Even morepreferably, they are alkali cations, for instance, Li⁺, Na⁺ or K⁺, oralkali earth cations, for instance, Mg⁺⁺, Ca⁺⁺ and the like. Sources ofthese cations are known to those of skill in the art and arecommercially available. Preferably, the source is a salt. Non-limitingexamples include CaCl₂, MgCl₂ and KCl and the like.

The term “ointment base” refers to a vehicle for substances intended forexternal application to the body. Generally, the base will comprise themain ingredient of a topically applied composition.

The term “glycoprotein” refers to a conjugated protein containing one ormore covalently linked carbohydrate residues.

The term “effective amount” refers to an amount sufficient to effectbeneficial or desired results. For purposes of this invention, aneffective amount of an antihistamine and/or a cation is an amountsufficient to inhibit, limit, repress, palliate or prevent the dischargeof nematocysts.

The term “final concentration” refers to the concentration of theingredient after the composition has been prepared, including, forinstance, the concentration of the composition after it has been placedin an aqueous environment.

The term “surrounding environment” refers to the aqueous medium whichsurrounds stinging marine organisms. It includes, for example, fishponds, aquariums, ocean enclosures and the like.

The present invention includes chemicals that inhibit nematocystdischarge. Without being bound to one theory, it is believed that thepresent invention acts to inhibit discharge by interfering withnematocyst discharge at three levels: (1) by reducing secretions ofstimulants from the skin; (2) by acting as an antagonist to thechemoreceptor trigger of discharge and (3) by interfering with the buildup of hydrostatic pressure required for discharge.

Inhibition of Nematocyst Discharge

Compositions of the present invention are effective at inhibitingdischarge of nematocysts. In one embodiment, the present inventionincludes compositions comprising an effective amount of antihistamine.In another embodiment, the present invention includes compositioncomprising an effective amount of antihistamine and an effective amountof at least one cation. Preferably, the antihistamine isdiphenhydramine, tripelennamine or cimetidine. These antihistamines arecommercially available from various suppliers. In a preferredembodiment, diphenhydramine is used. The antihistamine is added to afinal concentration of at least about 0.0005% (or 15 μM). Preferably,the final concentration is from about 0.0005% to about 2%. Morepreferably, the concentration is between about 0.001% and about 1%. Evenmore preferably, the concentration is between about 0.001% and 0.2%.Preferably, the concentration of cations is from about 50 mM to about 1M. More preferably, the concentration is from about 50 mM to about 500mM. Even more preferably, the concentration is from about 50 mM to about200 mM. Concentrations greater than 1 M are also likely to be effective.It is believed that higher concentrations of the antihistamine orcations would be effective, but in view of cost considerations and anypotential side-effects, the amounts specified above are preferred.

Hydrostatic pressure is the driving force for nematocyst discharge. Togenerate the needed pressure, bound cations (Ca⁺⁺, K⁺, Mg⁺⁺, NH₄ ⁺, Na⁺,and/or Co⁺⁺) are released from the internal nematocyst capsule. Forexample, the Ca⁺⁺ concentration in internal resting nematocysts is300-600 mM, this Ca⁺⁺ is bound to the PGA matrix. Releasing the Ca⁺⁺during nematocyst discharge increases the tonicity of the internalcapsule and transforms osmotic pressure into hydrostatic pressure. Ca⁺⁺concentration in the nematocyte cell, outside of the nematocyst capsule,and in sea water is 7 mM. When the Ca⁺⁺ concentration in the surroundingfluids of the capsule reaches 50 mM, the osmolarity of the nematocyst isreduced and discharge is less likely to occur. Because the cationsinvolved in nematocyst discharge may differ between species, the presentinvention encompasses adding a mixture of cations. In this way,inhibition can be effected for a broader range of species, for example,in both hydra and sea anemones. In another aspect, an effective amountof only one cation is used. Preferably, the cations used are Ca⁺⁺, K⁺,Na⁺⁺, Mn⁺⁺, Co⁺⁺, Mg⁺⁺, and Fe⁺⁺. Even more preferably, they are Ca⁺⁺,K⁺ and Mg⁺⁺. These cations can be obtained from various commercialvendors.

In yet another embodiment, compositions comprising an effective amountof at least one cation are provided. Preferably, the concentration ofcations is from about 50 mM to about 1 M. More preferably, theconcentration is from about 50 mM to about 500 mM. Even more preferably,the concentration is from about 50 mM to about 200 mM. Concentrationsgreater than 1 M are also likely to be effective. In a preferredembodiment, at least one cation is included in the ointment basedetailed in the following section.

In yet a further aspect, the invention includes adding the compositionsdescribed herein to the aqueous environment surrounding thenematocyst-containing organism. An effective amount of the compositionis added to the aqueous environment surrounding the nematocysts. Theconcentrations described above are preferable.

Topical Formulations

In another aspect, compositions of the present invention are formulatedfor topical application. Preferably, topical formulations containingcompositions within the present invention create physical barriers tominimize skin stimulant secretion and, are themselves, not stimulants.Formulations should be water-proof so as to be useful for swimmers anddivers. Ca⁺⁺ binding compound such as sodium citrate, EDTA, amino acidsand glycoproteins generate nematocysts discharge and are, therefore, notto be used. Examples of suitable carriers include, but are not limitedto, dextran, dextran sulfate, agarose, phosphatidyl ethanolamine,cholesterol, cholesterol palmitate, palmitic acid, oleic acid,lysolecitin, phosphatidyl choline, phosphatidyl serine, phosphatidylinositol, squalene, phenyl trimethicone, stearic acid, propylene glycolsterate, petrolatum, stearoxytrimethyl-silane, stearyl alcohol. Othercarriers are known to those in the art. Silicone polymers are preferredas they provide good barriers, are water-proof and do not stimulatenematocyst discharge.

Optionally, the compositions described herein may contain a knownsunscreen. Preferably, the sunscreens contain at least one UVA filterand at least one UVB filter. Oil-soluble UVB products include3-benzylidenecamphor derivatives, 4-aminobenzoic acid derivatives,esters of cinnamic acid, derivatives of benzophenone, esters ofbenzylidenemalonic acid. Water soluble UVA filters include salts of2-phenylbenzimidazole-5-sulphonic acid, sulphonic acid derivatives ofbenzophenones and sulphonic acid derivatives of 3-benzylidenecamphor.Effective amounts of sunscreens will be known to those of skill in theart. Preferably, sunscreens will comprise between about 0.1% and 30% byweight of the total preparation.

The following examples are intended to illustrate but not to limit thesubject invention.

EXAMPLES Example 1

Decrease in Nematocyst Discharge Using Antihistamine in the AqueousEnvironment

To initially test inhibition of nematocyst discharge, the following testwas performed. It is typical behavior for jellyfish or sea anemonetentacles to adhere to and paralyze newly hatched nauplii of Artemia sp.The adhesion and subsequent paralysis is characteristic of nematocystdischarge by the tentacles. Small 2 to 3 mm fragments of jellyfish orsea anemone tentacles were prepared and placed in a 12-well platecontaining filtered sea water. The tentacle fragments were washed bychanging the filtered sea water three times. One mL of filtered seawater was left in each well after the final wash. In 6 of the 12 wells,the antihistamine diphenhydramine hydrochloride was added to finalconcentration of 0.02%. The 6 wells without the antihistamine served ascontrols. Ten Artemia sp. were added to each well in a total volume ofbetween 50 and 100 uL of filtered sea water. After 2 minutes, the numberof attached or paralyzed Artemia in each well was measured. Results areshown in Table 1.

TABLE 1 Surrounding water No. paralyzed Artemia Total No. Artemia seawater + antihistamine 2 10 sea water + antihistamine 1 8 sea water +antihistamine 2 12 sea water + antihistamine 3 14 sea water +antihistamine 2 10 sea water + antihistamine 1 10 sea water alone 8 10sea water alone 9 13 sea water alone 9 11 sea water alone 7 10 sea wateralone 9 11 sea water alone 8 9

Taken together, these results show that 11 out of 64 Artemia wereparalyzed by nematocysts when antihistamine was added to theirenvironment. In contrast, 50 of 64 Artemia were paralyzed by nematocystswhen no antihistamine was present. Diphenhydramine significantlydecreased the number of Artemia paralyzed by nematocysts.

Example 2

Inhibition of Nematocyst Discharge Into Human Skin UsingTopically-applied Antihistamine

To test the ability of antihistamines to inhibit nematocyst discharge,an assay using human skin was performed as described herein.

1. Preparation of Skin Samples

Normal human skin (HS) was obtained from neonatal elective circumcision.The skin was immediately placed into sterile phosphate buffered saline(PBS) containing antibiotic/antimycotic. The tissue was frozen embeddedin Tissue-Tek™. The tissue was then sectioned into 50 μm sections usinga cryostat. The sections were attached to glass slides coated withpolylysine and maintained at −70° C. until time of use. All sectionswere examined to ensure that they were perpendicular to the plane of theepidernis, exposing cross-sections of epidermis, dermis andsub-cutaneous tissue.

2. Immunoassay to Detect Nematocyst Discharge

A polyclonal antibody specific for nematocyst toxin was prepared, asdescribed in Lotan et al. (1996), supra. Briefly, the HPLC fractioncontaining the toxin phospholipase A2 from the jellyfish Rhopilemanomadica was isolated and injected into rabbits as described inVaitukaitis, pp. 46-52, “Production of Antisera with Small Doses ofimmunogen: Multiple Intradermal Injections, ” in METHODS IN ENZYMOLOGY,VOL. 73, ed. Larson & Vunkis (Academic Press, New York, 1981). Serum wascollected from the rabbits after two injections, and the presence oftoxin examined on immunoblots.

The slides prepared and frozen as described above were thawed at roomtemperature. The slides were submersed in sea water for approximately 10seconds and then attached to jellyfish or sea anemones tentacles forapproximately 10 seconds. The tentacles were detached, the slides washedtwo times with PBS, and blocked using 5% Fetal Bovine serum (FBS), and0.1% Triton X100 in PBS (blocking solution). All washing and incubationsteps were conducted on a tilting shaker at room temperature. The slideswere incubated in 10 mL of blocking solution containing a 1:2,500dilution of the anti-toxin antibody for one hour. After incubation, theslides were washed five times for five minutes each wash with 100 mL of0.1% Triton X100 in PBS (Wash solution). The slides were then incubatedin 10 mL blocking solution containing a second antibody, Goat antirabbit FITC (Fluorescent marker) for 30 minutes. The excess PBS wasremoved by shaking the slides. One drop of glycerol was added to eachslide and the slides covered with a glass coverslip. The slides wereobserved under a conventional light microscope or under an ultraviolet(UV) microscope at least at 200× magnification.

3. Inhibition of Nematocyst discharge into Human Skin Samples

To monitor nematocyst discharge, three types of live jellyfish, Aureliaaurita, Pelagia noctiluca, and Chrysaora quinquecirrha and two speciesof sea anemone, Anthoplaura elegantissima and Corynactis californicawere used in this experiment.

Four frozen human skin slides were thawed at room temperature. Twoslides were submersed in glycerol (control) and the other two weresubmersed in glycerol containing 0.2% diphenhydramine. The slides wereplaced vertically on a glass dish to allow the glycerol to drip off. Theslides were then exposed to one of the jellyfish or sea anemone speciesnoted above and the number of nematocysts discharged was determinedusing immunohistochemical analysis. On the glycerol only slides, 135 andover 500 nematocysts were counted per slide. On the glycerol plusantihistamine (diphenhydramine) 10 and 52 nematocysts were counted.These results demonstrate that the antihistamine diphenhydramine iseffective at inhibiting nematocyst discharge into human skin samples.

To closely parallel the natural conditions under which jellyfish stinghumans, the experiment described above was performed at the Monterey BayAquarium with the Chrysaora quinquicirrha and Pelagia noctiluca speciesof jellyfish. A total of 35 human slides were submersed in control ortest substances and exposed to the jellyfish. Six untreated slides,seven slides submersed in glycerol, and seven submersed in glycerol plus0.2% diphenhydramine were exposed to each jellyfish species forapproximately 10 to 15 seconds. The human skin slides exposed toChrysaora quinquecirrha showed that diphenhydramine decreases nematocystdischarge by 2.5-5 fold. In Pelagia noctiluca significant reduction innematocyst discharged was observed. A ten-fold decrease in nematocystdischarge was observed with Aurelia (N=3 for the test and 3 for eachcontrol), however, a low number of nematocysts was discharged in thecontrol (less than 100) thus leading to less striking results ascompared with those obtained with the other species.

Example 3

Inhibition of Nematocyst Discharge using Antihistamine and Cations

Frozen human skin slides are thawed at room temperature and submersed inglycerol containing 0.2% diphenhydramine and 100 mM CaCl₂, KCl or MgCl₂.Control slides are submersed in glycerol only. The slides are placedvertically in a glass dish to allow the glycerol to drip off. The slideis then exposed to jellyfish or sea anemone for about 15 seconds. Thenumber of nematocysts that adhere to the skin sample is counted usingthe procedure described in Example 2.

Example 4

Inhibition of Myxozoan infection

Host recognition and attachment of parasites to their host organisms iseffected by nematocyst discharge. For example, the triactinomixon stageof infection recognizes and infects specific fish hosts by extrusion ofa polar capsule. Yokoyama, supra. The filaments of the polar capsulepenetrate the fish epidermis and anchor the parasites to its host.

To prevent parasitic infections, a final concentration of 0.02%antihistamine diphenhydramine hydrochloride and 100 mM of a cation,e.g., CaCl₂, MgCl₂, or KCl, is added to the fish pond or aquarium. Thecation is selected based on the cation that is normally found in thelumen of the polar capsule. Unlike cnidarian nematocysts, triactinomyxonare exposed to the surrounding environment. Yokoyama, supra havepreviously shown that the ionic strength of the aqueous environmentinterferes with the internal structure of the triactinomyxon. Thus, thepresent invention involves preventing fish infection by two mechanisms:(1) antihistamines to block recognition of the host and (2) cations toinactive the polar capsule.

Fish between 14 and 21 days old that are susceptible to triactinomyxonare exposed to an average of 10,000 waterborne triactinomyxon forapproximately 1 hour in a total volume of 100 mL of water. In testaquariums, the water contains 0.02% diphenhydramine hydrochloride and100 mM KCl, MgCl₂ or CaCl₂. Control aquariums have no antihistamine orcation. The level of triactinomyxon infection is determined bymicroscopy, or by immunoassay as described in Example 2. The sameconcentrations of antihistamine and cation can be used in larger scalefish ponds or aquariums.

Example 5

Inhibition of Nematocyst Discharge Into Human Skin UsingTopically-applied Cations

To test the ability of cations to inhibit nematocyst discharge, an assayusing human skin is performed as described herein.

1. Preparation of Skin Samples

Normal human skin (HS) is obtained from neonatal elective circumcision.The skin is immediately placed into sterile phosphate buffered saline(PBS) containing antibiotic/antimycotic. The tissue is frozen embeddedin Tissue-Tek™. The tissue is then sectioned into 50 μm sections using acryostat. The sections are attached to glass slides coated withpolylysine and maintained at −70° C. until time of use. All sections areexamined to ensure that they are perpendicular to the plane of theepidermis, exposing cross-sections of epidermis, dermis andsub-cutaneous tissue.

2. Immunoassay to Detect Nematocyst Discharge

A polyclonal antibody specific for nematocyst toxin is prepared, asdescribed in Lotan et al. (1996), supra. Briefly, the HPLC fractioncontaining the toxin phospholipase A2 from the jellyfish Rhopilemanomadica is isolated and injected into rabbits as described inVaitukaitis, pp. 46-52, “Production of Antisera with Small Doses ofimmunogen: Multiple Intradermal Injections, ” in METHODS IN ENZYMOLOGY,VOL. 73, ed. Larson & Vunkis (Academic Press, New York, 1981). Serum iscollected from the rabbits after two injections, and the presence oftoxin examined on immunoblots.

The slides prepared and frozen as described above are thawed at roomtemperature. The slides are submersed in sea water for approximately 10seconds and then attached to jellyfish or sea anemones tentacles forapproximately 10 seconds. The tentacles are detached, the slides washedtwo times with PBS, and blocked using 5% Fetal Bovine serum (FBS), and0.1% Triton X100 in PBS (blocking solution). All washing and incubationsteps are conducted on a tilting shaker at room temperature. The slidesare incubated in 10 mL of blocking solution containing a 1:2,500dilution of the anti-toxin antibody for one hour. After incubation, theslides are washed five times for five minutes each wash with 100 mL of0.1% Triton X100 in PBS (Wash solution). The slides are then incubatedin 10 mL blocking solution containing a second antibody, Goat antirabbit FITC (Fluorescent marker) for 30 minutes. The excess PBS isremoved by shaking the slides. One drop of glycerol is added to eachslide and the slides covered with a glass coverslip. The slides areobserved under a conventional light microscope or under an ultraviolet(UV) microscope at least at 200× magnification.

3. Inhibition of Nematocyst discharge into Human Skin Samples

To closely parallel the natural conditions underwhich jellyfish stinghumans, the experiment described herein are performed at the MontereyBay Aquarium with the Pelagia noctiluca, and Chrysaora quinquecirrhaspecies of jellyfish.

Thirty frozen human skin slides are thawed at room temperature. Tenuntreated slides, ten slides submersed in glycerol and ten slidessubmersed in glycerol containing 200 mM CaCl₂ plus 200 mM MgCl₂ areexposed to each jellyfish species for approximately 10 to 15 seconds.The slides are analyzed to determine the number of nematocystsdischarged using immunohistochemical analysis. The untreated slides andthe slides treated only with glycerol prior to exposure to the jellyfishshow equivalent results with an average of 500 nematocysts per sample.The slides treated with glycerol containing 200 mM CaCl plus 200 mM MgClshow a 5-fold decrease in the number of nematocysts, an average of 100per slide, compared to the untreated slides and those treated withglycerol only.

Example 6

Inhibition of Nematocyst Discharge Using Cations on Human Subjects

Three square centimeters of the left inner arm of a human subject iscovered with glycerol containing 200 mM CaCl₂ plus 200 mM MgCl₂. The armis submersed in sea water and after 5 seconds exposed to the fishingtentacles of Chrysaora quinquecirrha (sea nettle) for approximately 10to 15 seconds. The same experiment is repeated using the right armcovered with glycerol alone as a control. Five human subjects areexposed to Chrysaora quinquecirrha and 5 additional subjects are exposedto the fishing tentacles of Pelagia noctiluca. The degree of skin damagein each subject is evaluated 5 minutes and one hour after exposure.Seven of the ten subjects tested show no skin damage on the left armwhile three exhibit minor damage to the left arm. Moderate to severeskin damage is observed on the right arm of these subjects.

Example 7

Inhibition of Myxozoan Infection

To prevent parasitic infections, a final concentration of 200 mM of acation, e.g., CaCl₂, MgCl₂, or KCl, preferably at least two cations suchas CaCl₂, MgCl₂, is added to the fish pond or aquarium. The cation isselected based on the cation that is normally found in the lumen of thepolar capsule. Unlike cnidarian nematocysts, triactinomyxon are exposedto the surrounding environment. Yokoyama, supra have previously shownthat the ionic strength of the aqueous environment interferes with theinternal structure of the triactinomyxon. Thus, the present inventioninvolves preventing fish infection by two mechanisms: (1) antihistaminesto block recognition of the host and (2) cations to inactive the polarcapsule.

Fish between 14 and 21 days old that are susceptible to triactinomyxonare exposed to an average of 10,000 waterborne triactinomyxon forapproximately 1 hour in a total volume of 100 mL of water. In testaquariums, the water contains 200 mM KCl, MgCl₂ or CaCl₂, or preferably200 mM of both CaCl₂ and MgCl₂. Control aquariums have no cation. Thelevel of triactinomyxon infection is determined by microscopy, or byimmunoassay as described in Example 2. The same concentrations of cationcan be used in larger scale fish ponds or aquariums.

I claim:
 1. A composition for inhibiting the discharge of nematocysts orpolar capsules, consisting essentially of Ca⁺⁺ and Mg⁺⁺ in a vehiclesuitable for topical application.
 2. The composition according to claim1, wherein the vehicle comprises a lipid.
 3. The composition accordingto claim 1, wherein the vehicle comprises a silicone polymer.
 4. Thecomposition according to claim 1, wherein the nematocysts are dischargedfrom a stinging organism selected from the phylum consisting of Cnidariaand Myxozoa.
 5. The composition according to claim 1, wherein theconcentration of Ca⁺⁺ is 100 mM and wherein the concentration of Mg⁺⁺ is100 mM.
 6. A composition for inhibiting the discharge of nematocysts orpolar capsules, consisting essentially of Ca⁺⁺, Mg⁺⁺ and a sunscreen ina vehicle suitable for topical application.